Fixed-Bed Bioreactor With Constant-Flow Pump / Tubing System

ABSTRACT

We have modified a commercially-available adherent cell culture bioreactor in several ways to increase productivity of cultured cells, while decreasing contamination risk. We found that modifying a commercially-available adherent cell culture bioreactor to provide for slower cell culture medium flow unexpectedly and dramatically increases the productivity of the cultured adherent cells. We also developed a new sampling manifold configuration and new way of taking samples, to reduce contamination risk.

RELATED APPLICATIONS

This application asserts priority to U.S. provisional patent filing Ser.No. 62/322,651, filed 14 Apr. 2016, the contents of which are hereincorporated by reference.

GOVERNMENT INTEREST

None.

BACKGROUND

We have improved commercial-scale adherent cell culture by developing animproved bioreactor which provides a ˜50% increase in productivity vizprior art bioreactors, while also eliminating a source of contamination.

Commercially-available adherent cell culture bioreactors include, forexample, the laboratory-scale iCELLis™ Nano (commercially available fromPall Corporation, Cambridge Masachusetts) and the commercial scaleiCELLis™ 500 bioreactor, which provides a volume of up to 74 liters ofcell culture medium and a cell culture substrate of medical gradepolyester microfibers which provide up to 500 m² growth area availableto the cells. We improved the function of such commercial-scalebioreactors by first, defining the process steps in a small scale andthen scaling these up into a large scale. Pall Life Science, themanufacturer of the iCELLis™ brand of bioreactors, was recommendingre-circulation or perfusion as a feeding strategy. The feeding strategyin small (Nano™) scale was tested by re-circulation, and later optimizedby perfusion.

Perfusion is a process step where cells in a bioreactor are continuouslyfeed with a fresh medium at the same time removing equal amount of spendmedium, which enables the cell growth in high cell density (Vellinga etal. 2014). The perfusion rate can vary depending on the type of cellline used, the polypeptide product produced by those cells, the specificcell culture medium employed and the cell growth system used. Animportant aspect in the removal of the spent medium is also to removethe (possible toxic) metabolic side products from the cell culture.These side products may have a negative effect on cell viability, andfurther may impair the productivity of producer or host cells.

Options for perfusion include batch perfusion and fed-batch perfusion(where feeding of fresh medium is performed but no removal of spendmedium is performed). The fed-batch type approach is also are-circulation strategy, where the cell culture medium volume is onlymedium is perfused at a rate slower than the slowest pump output volumeavailable in commercially-available apparatus. To be able to get aproperly low medium perfusion rate for our purposes, we programmed thepumps to run for certain time interval, followed by an interval wherethe pumps did not pump at all.

Because of this need to vary pump output, we have seen in practice thatthe stock iCELLis™ 500 bioreactor Feed Out pump provided by themanufacturer is not capable of removing medium out from the bioreactorvessel in a well controlled manner. We thus becan to test the viabilityof lower-output pumps able to provide an adequately low output flow.

EXAMPLE 2—CONSTANT LOW-VELOCITY PUMPING

We then investigated whether the variable media flow used in the priorart might impact cell culture in some way. To do this, we replaced theprior art Feed In pump provided with the iCELLis™ 500 bioreactor with areplacement pump which was able to provide a lower output volume, about16.7 L/day. For a 25 liter capacity bioreactor vessel, this means themedia in the vessel would be exchanged once every 3½ to 4½ days, ratherthan the nearly twice a day typical in the prior art. This reduces theflow across e.g., a 100 m² substrate surface area from at least 42.3 Lper 100 m² per day to 16.7 L per 100 m² per day. This lower Feed In pumprate enabled us to for the first time to run the Feed In pumpconstantly, without periodic stoppages.

During these runs it was observed that the Feed Out pump provided by themanufacturer was not able to perform the removal of the media from thebioreactor vessel throughout the process as planned. We thereforesimilarly replaced the prior art Feed Out pump provided by themanufacturer with a lower-output pump.

We performed a commercial-scale manufacturing run, using recombinantadherent producer cells to produce a recombinant adenovirus bearing atransgene (useful for e.g., gene therapy), using in the adherent cellculture process a combination of the stock iCELLis™ 500 Feed In pump andour own lower-output Feed Out pump. Our method is generally advantageousin producing vector with any kind of transgene (including therapeutictransgenes and marker transgenes such as green fluorescent protein), orany other genetic element or nucleotide sequence (e.g., viral vectorcontaining RNA transgene, shRNA, IngRNA, eRNA etc.).

We also performed three commercial-scale manufacturing runs, wherein weused our own lower-output Feed Out pump and also replaced the stockiCELLis™ 500 high-output Feed In pump also with a lower-output pump. Wereceived significantly higher adenoviral production in the adherentproducer cells in each of these three runs. In these three runs, theproductivity of viral particles per cell had increased 49.4% as comparedto productivity using the prior art higher-output pumps.

When we changed the process to work with lower-output pumps, our viralproductivity per cell surprisingly increased 49.4%. Without intendingfor the legal coverage of our patent to be bound by any scientificcausal theory, this improvement may be due to feeding the bioreactorvessel constantly with fresh medium, thereby keeping stable the level ofnutrients in the media in contact with the cultured cells. For example,we have found that adherent cells are most productive when the mediaflow is substantially constant and slow enough to maintain in the cellculture medium a level of lactate of not more than about 1.6 gramslactate/liter of culture medium. Similarly, we have found that certainadherent producer cells are most productive when the concentration ofglucose in the culture media is maintained at between about 0.5 andabout 1.0 grams of glucose per liter of media. Other adherent producercells are most productive with a glucose concentration which is higher(e.g., at least about 2.9 grams per liter) but nonetheless maintained ata relatively constant concentration due to low-velocity butsubstantially constant media flow. Alternatively, this may be due to theslow but constant flushing away of unwanted cellular waste products fromthe cell surface. Alternatively, this could be due to avoiding thephysical shear stress placed on the cultured cells when using thehigher-velocity media flow required by prior art hardware.Alternatively, this increase could be due to slower media flow enablingeach producer cell a longer time to produce virus-like particles.Whatever the cause (or causes), we found that a slower, constant mediumflow surprisingly and significantly increased adherent producer cellproductivity.

This increase is particularly surprising in light of the fact thatsuspension cell culture (e.g., the CultiBag RM™ suspension cell culturebag, commercially available from Sartorius Corp., Cambridge Mass.)provides more-or-less constant media flow across the cell surface, yetsuspension cell culture can be as less productive than adherent cellculture.

Lower-output pumps may also be successfully used for inoculation of thehost cells into the adherent bioreactor vessel, for drawing samples ofthe cell culture media during cell growth, and for harvesting e.g.,culture media at the end of the cell growth. We have surprisingly foundthat using a lower-output pump to perform these functionscounter-intuitively makes each of those processes faster than with theprior art high-output pumps.

We claim:
 1. An adherent cell culture bioreactor comprising a substratefor adherent cell culture and a pump able to provide a constant-rateoutput of cell culture media of less than about 50 mL of media per 1,800cells per day.
 2. The adherent cell culture bioreactor of claim 1, thepump able to provide a minimum constant-rate output of not more thanabout 16.7 mL of media per 1,800 cells per day.
 3. The adherent cellculture bioreactor of claim 1, the substrate providing at least about 60m² of surface area for adherent cell culture.
 4. The adherent cellculture bioreactor of claim 3, the substrate providing at least about400 m² of surface area for adherent cell culture.
 5. The adherent cellculture bioreactor of claim 1, configured to contain at least about 20liters of cell culture media.
 6. A recombinant polypeptide produced byculturing adherent producer cells in the adherent cell bioreactor ofclaim
 1. 7. The recombinant polypeptide of claim 6, where thepolypeptide comprises virus.
 8. The recombinant polypeptide of claim 7,where the virus comprises a viral capsid containing a non-viraltransgene.
 9. A method for culturing adherent cells on a substratecomprising: a. obtaining the bioreactor of claim 1, and then b. addingto the bioreactor cell culture media and cells, and then c. culturingthe cells in adherent mode on the substrate, while circulating the cellculture media about the cells at a rate of less than about 50 mL ofmedia per 1,800 cells per day.
 10. The method of claim 9, the substrateproviding at least about 400 m² of surface area for adherent cellculture.
 11. The method of claim 9, the bioreactor configured to containat least about 20 liters of cell culture media.
 12. An adherent-cellbioreactor comprising a container containing a substrate for adherentcell culture and a pump configured to be able to provide a constant-rateoutput of cell culture medium at a pump output volume sufficient tomaintain at a relatively constant concentration in said cell culturemedium a parameter selected from the group consisting of: concentrationof lactate in said medium of not more than about 1.58 grains/liter;concentration of glucose in said medium of between about 0.5 and 1.0grams/liter; and concentration of glucose in said medium of not lessthan about 2.89 grams/liter.
 13. The adherent cell culture bioreactor ofclaim 12, configured to contain at least about 20 liters of cell culturemedia.
 14. The adherent cell culture bioreactor of claim 12, thesubstrate providing at least about 60 m² of surface area for adherentcell culture.
 15. The adherent cell culture bioreactor of claim 14, thesubstrate providing at least about 400 m² of surface area for adherentcell culture.
 16. The adherent cell bioreactor of claim 12, the pumpconfigured to be able to provide a constant-rate output of cell culturemedium at a pump output volume sufficient to maintain in said cellculture medium a concentration of lactate in said medium of not morethan about 1.58 grams/liter.
 17. The adherent cell bioreactor of claim12, the pump configured to be able to provide a constant-rate output ofcell culture medium at a pump output volume sufficient to maintain insaid cell culture medium a concentration of glucose in said medium ofbetween about 0.5 and 1.0 grams/liter;
 18. The adherent cell bioreactorof claim 12, the pump configured to be able to provide a constant-rateoutput of cell culture medium at a pump output volume sufficient tomaintain in said cell culture medium a concentration of glucose in saidmedium of not less than about 2.89 grams/liter.
 19. A recombinantpolypeptide produced by culturing adherent producer cells in theadherent cell bioreactor of claim
 12. 20. The recombinant polypeptide ofclaim 19, where the polypeptide comprises virus.
 21. The recombinantpolypeptide of claim 20, where the virus comprises a viral capsidcontaining a non-viral transgene.
 22. A method for culturing adherentcells on a substrate comprising: a. obtaining the bioreactor of claim12, and then b. adding to the bioreactor cell culture media and cells,and then c. culturing the cells in adherent mode on the substrate, whilecirculating the cell culture media about the cells at a rate to maintainin said cell culture medium a parameter selected from the groupconsisting of: concentration of lactate in said medium of not more thanabout 1.58 grains/liter; concentration of glucose in said medium ofbetween about 0.5 and 1.0 grams/liter; and concentration of glucose insaid medium of not less than about 2.89 grams/liter.
 23. The method ofclaim 22, the substrate providing at least about 60 m² of surface areafor adherent cell culture.
 24. The method of claim 23, the substrateproviding at least about 400 m² of surface area for adherent cellculture.
 25. The method of claim 22, the bioreactor configured tocontain at least about 20 liters of cell culture media.
 26. An adherentcell culture bioreactor providing a substrate for adherent cell growthand configured to contain cell culture media and having a pump able topump said media, the pump configured to be able to provide aconstant-rate output of media at an output volume of less than about 50mL media per m² substrate area per day.
 27. The adherent cell culturebioreactor of claim 26, configured to contain at least about 20 litersof cell culture media.
 28. The adherent cell culture bioreactor of claim26, the substrate providing at least about 60 m² of surface area foradherent cell culture.
 29. The adherent cell culture bioreactor of claim28, the substrate providing at least about 400 m² of surface area foradherent cell culture.
 30. The adherent cell culture bioreactor of claim26, wherein the pump is connected to the substrate via a tube having aninterior diameter sized to accommodate the pump output volume of fluidwithout the formation of air bubbles inside the tube.
 31. A recombinantpolypeptide produced by culturing adherent producer cells in theadherent cell bioreactor of claim
 26. 32. The recombinant polypeptide ofclaim 31, where the polypeptide comprises virus.
 33. The recombinantpolypeptide of claim 32, where the virus comprises a viral capsidcontaining a non-viral transgene.
 34. A method for culturing adherentcells on a substrate comprising: a. obtaining the bioreactor of claim26, and then b. adding to the bioreactor cell culture media and cells,and then c. culturing the cells in adherent mode on the substrate, whilecirculating the cell culture media about the cells at a rate of lessthan about 50 mL of media per m² substrate area per day.
 35. The methodof claim 34, the substrate providing at least about 60 m² of surfacearea for adherent cell culture.
 36. The method of claim 35, thesubstrate providing at least about 400 m² of surface area for adherentcell culture.
 37. The method of claim 34, the bioreactor configured tocontain at least about 20 liters of cell culture media.
 38. A methodcomprising: a. obtaining an adherent cell culture bioreactor having asubstrate for adherent cell culture and a first sampling tube having aninput end and an output end, the input end of the first sampling tube incommunication with the substrate whereby liquid can flow from thesubstrate into the input end of the first sampling tube, through thefirst sampling tube and exit the bioreactor at the output end of thefirst sampling tube; and then b. adding cell culture media to thebioreactor, seeding cells into the bioreactor and culturing cellsadherent to the substrate, and c. flowing the cell culture media throughthe first sampling tube, and then d. removing from the first samplingtube output end at least one removed portion of cell culture media; andthen e. permanently sealing the first sampling tube without returninginto the output end of the sampling tube a material amount of theremoved portion of cell culture media.
 39. The method of claim 38, theadherent cell culture bioreactor further comprising a second samplingtube having an input end and an output end, the input end incommunication with the substrate whereby liquid can flow from thesubstrate into the input end of the second sampling tube, through thesecond sampling tube and exit the bioreactor at the output end of thesampling tube; the method further comprising: f. flowing the cellculture media through the second sampling tube, and then g. removingfrom the second sampling tube output end a second removed portion ofcell culture media; and then h. permanently sealing the second samplingtube without returning into the output end of the second sampling tube amaterial amount of the removed cell culture media.
 40. The method ofclaim 38, wherein the bioreactor is configured to contain at least about20 liters of cell culture media.
 41. The method of claim 38, thesubstrate providing at least about 60 m² of surface area for adherentcell culture.
 42. The method of claim 39, the substrate providing atleast about 400 m² of surface area for adherent cell culture.
 43. Themethod of claim 39, wherein (b) the cells adherent to the substrateexpress a recombinant polypeptide.
 44. The recombinant polypeptideproduced by the method of claim
 43. 45. The recombinant polypeptide ofclaim 44, where the polypeptide comprises virus.
 46. The recombinantpolypeptide of claim 45, where the virus comprises a viral capsidcontaining a non-viral transgene.
 47. An adherent cell culturebioreactor comprising: a. a substrate for adherent cell culture inflowable communication with at least two pairs of closable samplingtubes, whereby cell culture media can flow from the substrate througheach sampling tube, and whereby each sampling tube may be asepticallyclosed after cell culture media flows from the substrate through thesampling tube, to prevent further media flow through the sampling tube;and b. at least one of each pair of sampling tubes configured to beaseptically attached to a detachable sampling container.
 48. Thebioreactor of claim 47, configured to contain at least about 20 litersof cell culture media.
 49. The bioreactor of claim 50, furthercomprising additional pairs of closable sampling tubes, at least one ofthe additional pairs of sampling tubes configured to be asepticallyattached to a detachable sampling container.
 50. The bioreactor of claim47, further comprising a third pair of closable sampling tubes, at leastone of the third pair of sampling tubes configured to be asepticallyattached to a detachable sampling container.
 51. The bioreactor of claim47, the substrate providing at least about 60 m² of surface area foradherent cell culture.
 52. The bioreactor of claim 51, the substrateproviding at least about 400 m² of surface area for adherent cellculture.
 53. A recombinant polypeptide produced by the bioreactor ofclaim
 47. 54. The recombinant polypeptide of claim 53, where thepolypeptide comprises virus.
 55. The recombinant polypeptide of claim54, where the virus comprises a viral capsid containing a non-viraltransgene.
 56. An adherent cell culture bioreactor vessel having asubstrate for adherent cell culture and a pump able to provide aconstant-rate output of cell culture media sufficient to exchange thecell culture media in the vessel less than once every about 3½ days. 57.The adherent cell culture bioreactor of claim 56, the pump able toprovide a constant-rate output of cell culture media sufficient toexchange the cell culture media in the vessel less than once every about4½ days.
 58. The adherent cell culture bioreactor of claim 56, thesubstrate providing at least about 60 m² of surface area for adherentcell culture.
 59. The adherent cell culture bioreactor of claim 58, thesubstrate providing at least about 400 m² of surface area for adherentcell culture.
 60. The adherent cell culture bioreactor of claim 56,configured to contain at least about 20 liters of cell culture media.61. A recombinant polypeptide produced by culturing adherent producercells in the adherent cell bioreactor of claim
 56. 62. The recombinantpolypeptide of claim 61, where the polypeptide comprises virus.
 63. Therecombinant polypeptide of claim 62, where the virus comprises a viralcapsid containing a non-viral transgene.
 64. A method for culturingadherent cells on a substrate comprising: a. obtaining the bioreactor ofclaim 56, and then b. adding to the bioreactor cell culture media andcells, and then c. culturing the cells in adherent mode on thesubstrate, while circulating the cell culture media about the cells at arate of less than about 50 mL of media per 1,800 cells per day.
 65. Themethod of claim 64, the substrate providing at least about 400 m² ofsurface area for adherent cell culture.
 66. The method of claim 64, thebioreactor configured to contain at least about 20 liters of cellculture media.